Chapter 5 Fixation and Processing

Dako IHC Staining Methods 5th Edition

Alcoholic Fixative Examples

Carnoy’s, Methacarn, Osmium Tetroxide, Acetone

Use of Alcoholic Fixatives

Have been used for IHC purposes in order to avoid loss of antigenicity caused by excessive formalin fixation, or for monoclonal antibodies that reacted against an epitope destroyed by formalin.

Osmium Tertroxide- Primarily used in Electron Micrography

Acetone- Use for fixation of frozen sections

Alcoholic Fixatives are typically used for?

Looking for lymphocytes using CD-specific markers, and in looking for immunoglobulins such as IgG, IgA, and IgM.

What are combination fixatives in relation to Alcoholic Fixatives?

These often combine Alcohol with formalin, calcium or other heavy metals, along with a buffering mixture in an effort to create a universal fixative for IHC standardization. These fixative solutions are commercially available, and formulations are not disclosed. Many of these fixatives also address RNA nad DNA fixation for genetic studies in fixed tissue.

Previously common reasons Frozen section was needed for IHC?

Lymph node studies because of the antigen destruction caused by formalin, and for studies of Axon nerve tissues where a thicker 10 um section was necessary.

What advancement has reduced the need for Frozen sections in IHC?

The introduction of antigen unmasking methods using heated water.

What are the current needs for Frozen sections?

Frozen sections are used when a quick examination is necessary, and also for direct and indirect immunofluorescence, in which formalin fixation could produce a weaker result.

Frozen section preparation for immunostaining

Frozen sections should be fixed in room temp acetone before storing (5 sec).

Before staining the sections should be fixed in 4 degree C Acetone for 10 min.

Then sections should be rehydrated in a buffer solution for 5 minutes before staining.

Blood smears can be fixed with?

Acetone or 10% NBF for 10 minutes

What precausetions should be taken before staining a blood smear?

If the sample contains a large numbers of erythrocytes the slides should be incubated in an endogenous peroxidase blocking solution to prevent the staining of endogenous peroxidase.

What are important factors for slide processing?

Fixation is the most important factor. If tissue is well fixed other variations in processing will have tittle affect on antigenicity.

No processes should raise the temperature above 60 degrees C, as this will result in severe loss of antigenicity.

What is the suggested use limit for xylene?

50 slides per 250 mL of xylene is the limit before xylene is no longer effective for deparaffination.